Hemostatic compositions, assemblies, systems, and methods employing particulate hemostatic agents formed from hydrophilic polymer foam such as chitosan

ABSTRACT

Improved hemostatic agents take the form of granules or particles that can be used to stanch, seal, or stabilize a site of hemorrhage, including a noncompressible hemorrhage.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/485,886 filed on Jul. 13, 2006, entitled “Hemostatic Compositions,Assemblies, Systems, And Methods Employing Particulate Hemostatic AgentsFormed From Hydrophilic Polymer Foam Such As Chitosan”; which is acontinuation in part of U.S. patent application Ser. No. 11/020,365filed on Dec. 23, 2004, entitled “Tissue Dressing Assemblies, Systems,and Methods Formed From Hydrophilic Polymer Sponge Structures Such asChitosan”; which is a continuation in part of U.S. patent applicationSer. No. 10/743,052, filed on Dec. 23, 2003, entitled “Wound Dressingand Method of Controlling Severe Life-Threatening Bleeding”; which is acontinuation in part of U.S. patent application Ser. No. 10/480,827,filed on Oct. 6, 2004, entitled “Wound Dressing and Method ofControlling Severe Life-Threatening Bleeding,” which was a nationalstage filing under 37 C.F.R. §371 of International Application No.PCT/U502/18757, filed on Jun. 14, 2002, which are each incorporatedherein by reference. U.S. patent application Ser. No. 11/485,886 alsoclaims the benefit of U.S. Provisional Application Ser. No. 60/698,734,filed Jul. 13, 2005, and entitled “Hemostatic Compositions, Assemblies,Systems, and Methods Employing Particulate Hemostatic Agents Formed fromHydrophilic Polymer Foam Such As Chitosan, which is incorporated hereinby reference.

FIELD OF THE INVENTION

The invention is generally directed to agents applied externally orinternally on a site of tissue injury or tissue trauma to amelioratebleeding, fluid seepage or weeping, or other forms of fluid loss.

BACKGROUND OF THE INVENTION

Hemorrhage is the leading cause of death from battlefield trauma and thesecond leading cause of death after trauma in the civilian community.Non-compressible hemorrhage (hemorrhage not readily accessible to directpressure, such as intracavity bleeding) contributes to the majority ofearly trauma deaths. Apart from proposals to apply a liquid hemostaticfoam and recombinant factor VIIa to the non-compressible bleeding sites,very little has been done to address this problem. There is a criticalneed to provide more effective treatment options to the combat medic forcontrolling severe internal hemorrhage such as intracavity bleeding.

Control of intracavity bleeding is complicated by many factors, chiefamong which are: lack of accessibility by conventional methods ofhemostatic control such as application of pressure and topicaldressings; difficulty in assessing the extent and location of injury;bowel perforation, and interferences caused by blood flow and pooling ofbodily fluids.

SUMMARY OF THE INVENTION

The invention provides improved hemostatic agents that can be used tostanch, seal, or stabilize a site of hemorrhage, including anoncompressible hemorrhage, such as at a site of intracavity bleeding.The invention provides rapid delivery of a safe and effective hemostaticagent to a general site of bleeding; enhanced promotion of strong clotformation at the site of bleeding; and ability (if necessary) to applytamponade over the field of injury. The invention also provides anenhanced rate of wound healing with reduced fibrotic adhesion andreduced opportunity for wound infection. The invention thereforeaddresses many of the significant issues related to current difficultiesin controlling hemorrhage including intracavitary hemorrhage andrecovery from these types of injury.

One aspect of the invention provides a hemostatic agent that can beapplied to a site of bleeding to stanch, seal, or stabilize the site,with our without the application of direct pressure or compression.

One aspect of the invention provides a hemostatic agent that takes theform of a granule or particle that can be applied to stanch, seal, orstabilize a hemorrhage, with our without the application of directpressure or compression.

One aspect of the invention provides a chitosan material in the form ofa granule or particle that can be applied to stanch, seal, or stabilizea hemorrhage, with our without the application of direct pressure orcompression.

One aspect of the invention provides a densified chitosan material inthe form of a granule or particle that can be applied to stanch, seal,or stabilize a hemorrhage, with our without the application of directpressure or compression.

One aspect of the invention provides a hemostatic agent matrix in theform of a granule or particle that carries within it dense chitosanbeads.

One aspect of the invention provides a hemostatic agent matrix in theform of a granule or particle that carries within it a polymer meshmaterial.

One aspect of the invention provides a composite of a hemostatic agentthat takes the form of a granule or particle interspersed with strips ofpieces of a polymer mesh material that can be applied together tostanch, seal, or stabilize a hemorrhage, with our without theapplication of direct pressure or compression.

One aspect of the invention provides a hemostatic agent that takes theform of a granule or particle that can be applied within a polymer meshsocklet to stanch, seal, or stabilize a hemorrhage, with our without theapplication of direct pressure or compression.

One aspect of the invention provides a chitosan material in the form ofa granule or particle that carries within it dense chitosan beads.

One aspect of the invention provides a chitosan material in the form ofa granule or particle that carries within it a polymer mesh material.

One aspect of the invention provides a composite of a chitosan materialthat takes the form of a granule or particle interspersed with strips ofpieces of a polymer mesh material that can be applied together tostanch, seal, or stabilize a hemorrhage, with our without theapplication of direct pressure or compression.

One aspect of the invention provides a hemostatic agent that takes theform of a granule or particle that can be applied within a polymer meshsocklet to stanch, seal, or stabilize a hemorrhage, with our without theapplication of direct pressure or compression.

One aspect of the invention provides methods of treat bleeding using thematerials having the technical features described.

One aspect of the invention provides methods of treat intracavitybleeding using the materials having the technical features described.

One aspect of the invention provides a granular hemostatic material thatis obtained from controlled grinding of deproteinized, and optionallyalso fully or partially decalcified, crustacean shell material, which isthen partially (30%) or near fully (80-85%) deacetylized.

Other features and advantages of the invention shall be apparent basedupon the accompanying description, drawings, and listing of keytechnical features.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic anatomic view of an intracavity site ofnoncompressible hemorrhage, into which a hemostatic agent has beenapplied to stanch, seal, or stabilize the site.

FIG. 1B is an enlarged view of the hemostatic agent shown in FIG. 1A,showing the granules or particles that comprise the agent.

FIG. 2 is a further enlarged view of the granules or particles shown inFIG. 1B.

FIG. 3 is a schematic flow chart view of a process of manufacturing thegranules or particles shown in FIG. 2 from a chitosan material.

FIG. 4 shows an alternate step in the manufacturing process shown inFIG. 3, in which dense beads of chitosan material are added to thegranules or particles.

FIG. 5 shows a granule or particle that is formed that contains densebeads of chitosan material.

FIG. 6 shows an alternate step in the manufacturing process shown inFIG. 3, in which strips of a polymer mesh material are added to thegranules or particles.

FIG. 7 shows a granule or particle that is formed that contains apolymer mesh material.

FIG. 8 shows a composite hemostatic agent comprising hemostatic granulesor particles mixed with strips of polymer mesh material.

FIG. 9 shows a bolus of the granules or particles shown in FIG. 2 or 4or 7 contained for delivery in a socklet of polymer mesh material.

FIG. 10 shows one way of delivering the bolus of the granules orparticles shown in FIG. 9 in the socklet of polymer mesh material to aninjury site.

FIGS. 11A and 11B show a way of delivering a bolus of the granules orparticles shown in FIG. 2 or 4 or 7 into a releasable polymer meshsocklet at an injury site.

FIG. 12 is an alternative way of delivering a bolus of the granules orparticles shown in FIG. 2 or 4 or 7 to an injury site without use of acontainment socklet or the like.

DETAILED DESCRIPTION

Although the disclosure hereof is detailed and exact to enable thoseskilled in the art to practice the invention, the physical embodimentsherein disclosed merely exemplify the invention, which may be embodiedin other specific structure. While the preferred embodiment has beendescribed, the details may be changed without departing from theinvention, which is defined by the claims. For the purpose ofillustration, the invention is disclosed in the context of treating anoncompressible hemorrhage. It should be appreciated that the inventionis generally capable of treating any hemorrhage, with or without theapplication of pressure.

I. HEMOSTATIC AGENT A. Overview

FIG. 1A shows a site 10 of an intracavity abdominal injury, where severeinternal bleeding will occur if steps are not taken to stanch, seal, orstabilize the site. The site 10 is the location of a noncompressiblehemorrhage, meaning that the hemorrhage is not readily accessible todirect pressure.

As shown in FIGS. 1A and 1B, a hemostatic agent 12 that embodies thefeatures of the invention has been applied to stanch, seal, or stabilizethe site 10 without the application of direct pressure or compression.The agent 12 takes the form of discrete particles 14 of a biodegradablehydrophilic polymer (best shown in FIG. 1B and FIG. 2).

The polymer of which the particles 14 are formed has been selected toinclude a biocompatible material that reacts in the presence of blood,body fluid, or moisture to become a strong adhesive or glue. Desirably,the polymer from which the particles 14 are formed also desirablypossess other beneficial attributes, for example, anti-bacterial and/oranti-microbial anti-viral characteristics, and/or characteristics thataccelerate or otherwise enhance the body's defensive reaction to injury.The polymer material comprising the particles 14 has desirably beendensified or otherwise treated to make the particles 14 resistant todispersal away from the site 10 by flowing blood and/or other dynamicconditions affecting the site 10.

The agent 12 thereby serves to stanch, seal, and/or stabilize the site10 against bleeding, fluid seepage or weeping, or other forms of fluidloss. The agent 12 also desirably forms an anti-bacterial and/oranti-microbial and/or anti-viral protective barrier at or surroundingthe tissue treatment site 10. The agent 12 can applied as temporaryintervention to stanch, seal, and/or stabilize the site 10 on an acutebasis. The agent 12 can also be augmented, as will be described later,to make possible more permanent internal use.

B. The Hemostatic Particles

The particles 14 shown in FIG. 2 may comprise a hydrophilic polymerform, such as a polyacrylate, an alginate, chitosan, a hydrophilicpolyamine, a chitosan derivative, polylysine, polyethylene imine,xanthan, carrageenan, quaternary ammonium polymer, chondroitin sulfate,a starch, a modified cellulosic polymer, a dextran, hyaluronan orcombinations thereof. The starch may be of amylase, amylopectin and acombination of amylopectin and amylase.

In a preferred embodiment, the biocompatible material of the particles14 comprises a non-mammalian material, which is most preferablypoly[β-(1→4)-2-amino-2-deoxy-D-glucopyranose, which is more commonlyreferred to as chitosan. The chitosan selected for the particles 14preferably has a weight average molecular weight of at least about 100kDa, and more preferably, of at least about 150 kDa. Most preferably,the chitosan has a weight average molecular weight of at least about 300kDa.

In forming the particles 14, the chitosan is desirably placed intosolution with an acid, such as glutamic acid, lactic acid, formic acid,hydrochloric acid and/or acetic acid. Among these, hydrochloric acid andacetic acid are most preferred, because chitosan acetate salt andchitosan chloride salt resist dissolution in blood, whereas chitosanlactate salt and chitosan glutamate salt do not. Larger molecular weight(Mw) anions disrupt the para-crystalline structure of the chitosan salt,causing a plasticization effect in the structure (enhanced flexibility).Undesirably, they also provide for rapid dissolution of these larger Mwanion salts in blood.

One preferred form of the particles 14 comprises an “uncompressed”chitosan acetate matrix of density less than 0.035 g/cm³ that has beenformed by freezing and lyophilizing a chitosan acetate solution, whichis then densified by compression to a density of from 0.6 to 0.5 g/cm³,with a most preferred density of about 0.25 to 0.5 g/cm³. This chitosanmatrix can also be characterized as a compressed, hydrophilic spongestructure. The densified chitosan matrix exhibits all of theabove-described characteristics deemed to be desirable. It alsopossesses certain structural and mechanical benefits that lendrobustness and longevity to the matrix during use, as will be describedin greater detail later.

After formation in the manner just described, the sponge structure isgranulated, e.g., by a mechanical process, to a desired particlediameter, e.g., at or near 0.9 mm.

The chitosan matrix from which the particles 14 are formed presents arobust, permeable, high specific surface area, positively chargedsurface. The positively charged surface creates a highly reactivesurface for red blood cell and platelet interaction. Red blood cellmembranes are negatively charged, and they are attracted to the chitosanmatrix. The cellular membranes fuse to chitosan matrix upon contact. Aclot can be formed very quickly, circumventing immediate need forclotting proteins that are normally required for hemostasis. For thisreason, the chitosan matrix is effective for both normal as well asanti-coagulated individuals, and as well as persons having a coagulationdisorder like hemophilia. The chitosan matrix also binds bacteria,endotoxins, and microbes, and can kill bacteria, microbes, and/or viralagents on contact. Furthermore, chitosan is biodegradable within thebody and is broken down into glucosamine, a benign substance.

C. Manufacture of the Hemostatic Particles

A desirable methodology for making the particles 14 will now bedescribed. This methodology is shown schematically in FIG. 3. It shouldbe realized, of course, that other methodologies can be used.

1. Preparation of a Chitosan Solution

The chitosan used to prepare the chitosan solution preferably has afractional degree of deacetylation greater than 0.78 but less than 0.97.Most preferably the chitosan has a fractional degree of deacetylationgreater than 0.85 but less than 0.95. Preferably the chitosan selectedfor processing into the matrix has a viscosity at 25° C. in a 1%(w/w)solution of 1%(w/w) acetic acid (AA) with spindle LVI at 30 rpm, whichis about 100 centipoise to about 2000 centipoise. More preferably, thechitosan has viscosity at 25° C. in a 1%(w/w) solution of 1%(w/w) aceticacid (AA) with spindle LVI at 30 rpm, which is about 125 centipoise toabout 1000 centipoise. Most preferably, the chitosan has viscosity at25° C. in a 1%(w/w) solution of 1%(w/w) acetic acid (AA) with spindleLV1 at 30 rpm, which is about 400 centipoise to about 800 centipoise.

The chitosan solution is preferably prepared at 25° C. by addition ofwater to solid chitosan flake or powder and the solid dispersed in theliquid by agitation, stirring or shaking. On dispersion of the chitosanin the liquid, the acid component is added and mixed through thedispersion to cause dissolution of the chitosan solid. The rate ofdissolution will depend on the temperature of the solution, themolecular weight of the chitosan and the level of agitation. Preferablythe dissolution step is performed within a closed tank reactor withagitating blades or a closed rotating vessel. This ensures homogeneousdissolution of the chitosan and no opportunity for high viscosityresidue to be trapped on the side of the vessel. Preferably the chitosansolution percentage (w/w) is greater than 0.5% chitosan and less than2.7% chitosan. More preferably the chitosan solution percentage (w/w) isgreater than 1% chitosan and less than 2.3% chitosan. Most preferablythe chitosan solution percentage is greater than 1.5% chitosan and lessthan 2.1% chitosan. Preferably the acid used is acetic acid. Preferablythe acetic acid is added to the solution to provide for an acetic acidsolution percentage (w/w) at more than 0.8% and less than 4%. Morepreferably the acetic acid is added to the solution to provide for anacetic acid solution percentage (w/w) at more than 1.5% (w/w) and lessthan 2.5%.

The structure or form producing steps for the chitosan matrix aretypically carried out from solution and can be accomplished employingtechniques such as freezing (to cause phase separation), non-solvent dieextrusion (to produce a filament), electro-spinning (to produce afilament), phase inversion and precipitation with a non-solvent (as istypically used to produce dialysis and filter membranes) or solutioncoating onto a preformed sponge-like or woven product. In the case offreezing, where two or more distinct phases are formed by freezing(typically water freezing into ice with differentiation of the chitosanbiomaterial into a separate solid phase), another step is required toremove the frozen solvent (typically ice), and hence produce thechitosan matrix 12 without disturbing the frozen structure. This may beaccomplished by a freeze-drying and/or a freeze substitution step. Thefilament can be formed into a non-woven sponge-like mesh by non-wovenspinning processes. Alternately, the filament may be produced into afelted weave by conventional spinning and weaving processes. Otherprocesses that may be used to make the biomaterial sponge-like productinclude dissolution of added porogens from a solid chitosan matrix orboring of material from said matrix.

2. Degassing the Aqueous Chitosan Solution

Preferably (see FIG. 3, Step B), the chitosan biomaterial 16 is degassedof general atmospheric gases. Typically, degassing is removingsufficient residual gas from the chitosan biomaterial so that, onundergoing a subsequent freezing operation, the gas does not escape andform unwanted large voids or large trapped gas bubbles in the subjectwound dressing product. The degassing step may be performed by heating achitosan biomaterial, typically in the form of a solution, and thenapplying a vacuum thereto. For example, degassing can be performed byheating a chitosan solution to about 45° C. immediately prior toapplying vacuum at about 500 mTorr for about 5 minutes while agitatingthe solution.

In one embodiment, certain gases can be added back into the solution tocontrolled partial pressures after initial degassing. Such gases wouldinclude but are not limited to argon, nitrogen and helium. An advantageof this step is that solutions containing partial pressures of thesegases form micro-voids on freezing. The microvoid is then carriedthrough the sponge as the ice-front advances. This leaves a well-definedand controlled channel that aids sponge pore interconnectivity.

3. Freezing the Aqueous Chitosan Solution

Next (see FIG. 3, Step C), the chitosan biomaterial 16—which istypically now in acid solution and degassed, as described above—issubjected to a freezing step. Freezing is preferably carried out bycooling the chitosan biomaterial solution supported within a mold andlowering the solution temperature from room temperature to a finaltemperature below the freezing point. More preferably this freezing stepis performed on a plate freezer whereby a thermal gradient is introducedthrough the chitosan solution in the mold by loss of heat through theplate cooling surface. Preferably this plate cooling surface is in goodthermal contact with the mold. Preferably the temperature of thechitosan solution and mold before contact with the plate freezer surfaceare near room temperature. Preferably the plate freezer surfacetemperature is not more than −10° C. before introduction of themold+solution. Preferably the thermal mass of the mold+solution is lessthan the thermal mass of the plate freezer shelf+heat transfer fluid.Preferably the molds are formed from, but are not limited to, a metallicelement such as iron, nickel, silver, copper, aluminum, aluminum alloy,titanium, titanium alloy, vanadium, molybdenum, gold, rhodium,palladium, platinum and/or combinations thereof. The molds may also becoated with thin, inert metallic coatings such as titanium, chromium,tungsten, vanadium, nickel, molybdenum, gold and platinum in order toensure there is no reaction with the acid component of the chitosansolution and the chitosan salt matrix. Thermally insulating coatings orelements may be used in conjunction with the metallic molds to controlheat transfer in the molds. Preferably the mold surfaces do not bindwith the frozen chitosan solution. The inside surface of the mold ispreferably coated with a thin, permanently-bound, fluorinated releasecoating formed from polytetrafluoroethylene (Teflon), fluorinatedethylene polymer (FEP), or other fluorinated polymeric materials.Although coated metallic molds are preferable, thin walled plastic moldscan be a convenient alternative for supporting the solution. Suchplastic molds would include, but not be limited to, molds prepared byinjection molding, machining or thermoforming from polyvinylchloride,polystyrene, acrylonitrile-butadiene

styrene copolymers, polyesters, polyamides, polyurethanes andpolyolefins. An advantage of the metallic molds combined with localplacement of thermally insulating elements is that they also provideopportunity for improved control of heat flow and structure within thefreezing sponge. This improvement in heat flow control results fromlarge thermal conductivity differences between thermally conducting andthermally insulating element placements in the mold.

Freezing of the chitosan solution in this way enables the preferredstructure of the agent 12 to be prepared.

The plate freezing temperature affects the structure and mechanicalproperties of the final chitosan matrix 16. The plate freezingtemperature is preferably not higher than about −10° C., more preferablynot more than about −20° C., and most preferably not more than about−30° C. When frozen at −10° C., the structure of the uncompressedchitosan matrix 16 is very open and vertical throughout the open spongestructure. When frozen at −25° C., the structure of the uncompressedchitosan matrix 12 is more closed, but it is still vertical. When frozenat −40° C., the structure of the uncompressed chitosan matrix 16 isclosed and not vertical. Instead, the chitosan matrix 16 comprises moreof a reinforced, inter-meshed structure. The adhesive/cohesive sealingproperties of the chitosan matrix 16 are observed to improve as lowerfreezing temperatures are used. A freezing temperatures of about −40° C.forms a structure for the chitosan matrix 16 having superioradhesive/cohesive properties.

During the freezing step, the temperature may be lowered over apredetermined time period. For example, the freezing temperature of achitosan biomaterial solution may be lowered from room temperature to−45° C. by plate cooling application of a constant temperature coolingramp of between about −0.4° C./mm to about −0.8° C./mm for a period ofabout 90 minutes to about 160 minutes.

4. Freeze Drying the Chitosan/Ice Matrix

The frozen chitosan/ice matrix desirably undergoes water removal fromwithin the interstices of the frozen material (see FIG. 3, Step D). Thiswater removal step may be achieved without damaging the structuralintegrity of the frozen chitosan biomaterial. This may be achievedwithout producing a liquid phase, which can disrupt the structuralarrangement of the ultimate chitosan matrix 16. Thus, the ice in thefrozen chitosan biomaterial passes from a solid frozen phase into a gasphase (sublimation) without the formation of an intermediate liquidphase. The sublimated gas is trapped as ice in an evacuated condenserchamber at substantially lower temperature than the frozen chitosanbiomaterial.

The preferred manner of implementing the water removal step is byfreeze-drying, or lyophilization. Freeze-drying of the frozen chitosanbiomaterial can be conducted by further cooling the frozen chitosanbiomaterial. Typically, a vacuum is then applied. Next, the evacuatedfrozen chitosan material may be gradually heated.

More specifically, the frozen chitosan biomaterial may be subjected tosubsequent freezing preferably at about −15° C., more preferably atabout −25° C., and most preferably at about −45° C., for a preferredtime period of at least about 1 hour, more preferably at least about 2hour, and most preferably at least about 3 hour. This step can befollowed by cooling of the condenser to a temperature of less than about−45° C., more preferably at about −60° C., and most preferably at about−85° C. Next, a vacuum in the amount of preferably at most about 100mTorr, more preferably at most about 150 mTorr, and most preferably atleast about 200 mTorr, can be applied. The evacuated frozen chitosanmaterial can be heated preferably at about −25° C., more preferably atabout −15° C., and most preferably at about −10° C., for a preferredtime period of at least about 1 hour, more preferably at least about 5hour, and most preferably at least about 10 hour.

Further freeze drying, maintaining vacuum pressure at near 200 mTorr, isconducted at a shelf temperature of about 20° C., more preferably atabout 15° C., and most preferably at about 10° C., for a preferred timeperiod of at least about 36 hours, more preferably at least about 42hours, and most preferably at least about 48 hours.

5. Densification of the Chitosan Matrix

The chitosan matrix 16 before densification (density near 0.03 g/cm′)will be called an “uncompressed chitosan matrix.” This uncompressedmatrix is ineffective in stanching bleeding since it rapidly dissolvesin blood and has poor mechanical properties. The chitosan biomaterial isnecessarily compressed (see FIG. 3, Step E). Compression loading normalto the hydrophilic matrix polymer surface with heated platens can beused to compress the dry “uncompressed” chitosan matrix 16 to reduce thethickness and increase the density of the matrix. The compression step,which will sometimes be called in shorthand “densification,”significantly increases adhesion strength, cohesion strength anddissolution resistance of the chitosan matrix 12. Appropriately frozenchitosan matrices 16 compressed above a threshold density (close to 0.1g/cm3) do not readily dissolve in flowing blood at 37° C.

The compression temperature is preferably not less than about 60° C.,more preferably it is not less than about 75° C. and not more than about85° C.

The densified chitosan biomaterial is next preferably preconditioned byheating chitosan matrix 16 in an oven to a temperature of preferably upto about 75° C., more preferably to a temperature of up to about 80° C.,and most preferably to a temperature of preferably up to about 85° C.(FIG. 3, Step F). Preconditioning is typically conducted for a period oftime up to about 0.25 hours, preferably up to about 0.35 hours, morepreferably up to about 0.45 hours, and most preferably up to about 0.50hours. This pre-conditioning step provides further significantimprovement in dissolution resistance with a small cost in a 20-30% lossof adhesion properties.

A backing may be secured to one side of the chitosan matrix 16 tofacilitate further handling. The backing can be attached or bonded bydirect adhesion with a top layer of chitosan matrix 16. Alternatively,an adhesive such as 3M 9942 Acrylate Skin Adhesive, or fibrin glue, orcyanoacrylate glue can be employed.

6. Granulation of the Densified Chitosan Matrix

Matrix 16 is granulated, e.g., by a mechanical process to a desiredparticle diameter, e.g., at or near about 0.9 mm. Simple mechanicalgranulation of the chitosan matrix 16 through a suitable mechanicaldevice 18 (as shown in FIG. 3, Step G) can be used to prepare chitosansponge particles 14 of close to 0.9 mm in diameter. Other granulationmethodologies can be used. For example, off the shelf stainless steelgrinding/granulating laboratory/food processing equipment can be used.More robust, purpose designed, and more process-controlled systems canalso be used.

Granulation of the chitosan matrix 16 can be conducted under ambienttemperature or liquid nitrogen temperature conditions.

Preferably, a well-defined particle size distribution of particlegranulate is prepared. The particle size distribution can becharacterized using, e.g., Leica ZP6 APO stereomicroscope and ImageAnalysis MC software.

7. Sterilization

The desired weight volume of particles 14 can be subsequently packagedin a pouch 20, which is desirably purged with an inert gas such aseither argon or nitrogen gas, evacuated and heat sealed. The pouch 20acts to maintain interior contents sterility over an extend time (atleast 24 months) and also provides a very high barrier to moisture andatmospheric gas infiltration over the same period.

After pouching, the particles 14 are desirably subjected to asterilization step (see FIG. 3, Step H). The particles 14 can besterilized by a number of methods. For example, a preferred method is byirradiation, such as by gamma irradiation, which can further enhance theblood dissolution resistance, the tensile properties and the adhesionproperties of the wound dressing. The irradiation can be conducted at alevel of at least about 5 kGy, more preferably a least about 10 kGy, andmost preferably at least about 15 kGy.

D. Altering the Properties of the Hemostatic Particles

The properties of the chitosan matrix 16 and thus of the particles 14formed from it may be further optimized to provide for improvedhemostatic performance to control non-compressible hemorrhage.

1. The Chitosan Salt Composition

For example, the composition of the chitosan salt can be optimized forpromotion of rapid clotting. It has been discovered that chitosan with ahigh degree of deacetylation and high molecular weight more readilyproduces rapid clotting than chitosan with lower degree ofdeacetylation. It has also been discovered that salts of acetic, lacticand glycolic acids provide for this enhanced clot formation when certainlevels of other adjuvants, e.g., iso-propyl alcohol, are present.

The local promotion of clotting can be augmented by adjusting thecomposition of the chitosan matrix 16 accordingly, e.g. by providing arange of high degree of deacetylation chitosan and high molecular weightmatrices 16 of different density, of different acid (lactic, glycolic,acetic) with different concentrations of adjuvants such as iso-propylalcohol. The matrices 16 can be granulated by a mechanical process,pouched, and sterilized prior to use in the manner previously described.

2. Homogeneous Mixing of the Chitosan Foam with Dense Chitosan Beads

Chitosan beads 22 (shown in FIG. 4) of controlled diameter can beprepared by flow mixing of a chitosan acid solution and a polyanionsolution (such as an alginate) across an oscillating electric field.After neutralization and drying, the beads 22 form small hard densespheres that are relatively insoluble. As shown in FIG. 4, addition andhomogeneous mixing of a significant fraction of these spherical beads 22to a viscous chitosan solution 16 immediately prior to the freezing step(as above described) will result in a homogeneous dispersion of beadsthrough the lyophilized foam sponge. In sufficient numbers these beads22 will provide for a high density core in the center of the foamgranulate particle 14, as FIG. 5 shows. The high density bead coreformed by the beads 22 assists in more local application of the beadedparticles 14 to a bleeding injury.

3. Mesh-Reinforced Particles

The interior of the particles 14 can be reinforced by the inclusion ofsmall strips or pieces of a bioresorbable polymer mesh material 24 (asshown in FIGS. 6 and 7). These strips of mesh material 24 can be addedto the viscous chitosan solution 16 immediately before the freezing step(as FIG. 6 shows). Alternatively (as FIG. 7 shows), loose small stripsor pieces of a bioresorbable polymer mesh material 24 can be added aftergranulation and prior to pouching and sterilization. In thisarrangement, the strips or pieces of mesh material 24 reside between theindividual particles 14 contained within the pouch 22 (as shown in FIG.8).

The presence of the mesh material 24 enhances hemostasis by overallreinforcement of the complex composite of chitosan granule particle 14,blood, and the mesh material 24.

The composition of the mesh material 24 can vary. It is believed that amesh formed from poly-4-hydroxy butyrate (TephaFLEXX Materialmanufactured by Tepha Inc.) is desirable. This material is abiosynthetic absorbable polyester produced through a fermentationprocess rather than by chemical synthesis. It can generally be describedas a strong, pliable thermoplastic with a tensile strength of 50 MPa,tensile modulus of 70 MPa, elongation to break of −1000%, and hardness(Shore D) of 52.8. Upon orientation the tensile strength increasesapproximately 10-fold (to a value about 25% higher than commercialabsorbable monofilament suture materials such as PDSII™).

Despite its biosynthesis route, the structure of the polyester is verysimple, and closely resembles the structures of other existing syntheticabsorbable biomaterials used in medical applications. The polymerbelongs to a larger class of materials called polyhydroxyalkanoates(PHAs) that are produced in nature by numerous microorganisms. In naturethese polyesters are produced as storage granules inside cells, andserve to regulate energy metabolism. They are also of commercialinterest because of their thermoplastic properties, and relative ease ofproduction. Tepha, Inc. produces the TephaFLEXTh biomaterial for medicalapplications using a proprietary transgenic fermentation processspecifically engineered to produce this homopolymer. The TephaFT.F.X™hiomateria] production process utilizes a genetically engineeredEscherichia coli K12 microorganism that incorporates new biosyntheticpathways to produce the polymer. The polymer accumulates inside thefermented cells during fermentation as distinct granules, and can thenbe extracted at the end of the process in a highly pure form. Thebiomaterial has passed tests for the following: cytotoxicity;sensitization; irritation and intracutaneous reactivity;hemocompatibility; endotoxin; implantation (subcutaneous andintramuscular); and USP Class VI. In vivo, the TephaFLEX™ biomaterial ishydrolyzed to 4-hydroxybutyrate, a natural human metabolite, presentnormally in the brain, heart, lung, liver, kidney, and muscle. Thismetabolite has a half-life of just 35 minutes, and is rapidly eliminatedfrom the body (via the Krebs cycle) primarily as expired carbon dioxide.

Being thermoplastic, the TephaFLEX™ biopolymer can be converted into awide variety of fabricated forms using traditional plastics processingtechnologies, such as injection molding or extrusion. Melt extrudedfibers made from this novel absorbable polymer are at least 30%stronger, significantly more flexible and retain their strength longerthan the commercially available absorbable monofilament suturematerials. These properties make the TephaFLEX™ biopolymer an excellentchoice for construction of a hemostatic dressing for controllingintracavity hemorrhage.

The TephaFLEXTh biomaterial can be processed into fibers and fabricssuitable for use as an absorbable sponge.

E. Delivery of the Particles

To provide for enhanced local delivery and potentially some pressurecompaction (tamponade) of the encased granulate against the wound, thechitosan granulate particles 14 can be desirable housed for deliverywithin an open mesh socklet or bag 26 (see FIG. 9) The socket 26 can bemade, e.g., from a TephaFLEX biomaterial above described.

The mesh of the socklet 26 is sufficiently open to allow for thechitosan granulate particles 14 to protrude out of the socklet 26, butnot so open that granulate particles 14 could be flushed away by flowingblood through the mesh. The socklet 26 supports the chitosan granulateparticles 14 during and after delivery and allows a more directedapplication of a bolus of the granulate particles 14. The mesh socklet26 should be sufficiently open to allow protrusion of chitosan particles14 at the outer surface of the bolus from its outside surface withoutloss of individual chitosan granule particles 14. The mechanicalproperties of the mesh socklet 26 are sufficient to allow localapplication of pressure over its surface without tearing or breaking.

The tamponade of a socklet 26 filled with the particles 14 can beapplied, e.g., through a cannula 28 (see FIG. 10) by use of tamp 34 toadvance the socklet 26 through the cannula 28 to the injury site 10.Multiple socklets 26 can be delivered in sequence through the cannula28, if required. Alternatively, a caregiver can manually insert one ormore of the socklets 26 into the treatment site 10 through a surfaceincision.

Alternatively, as FIGS. 11A and 11B show, a mesh socklet 30 can bereleasably attached to the end of a cannula 28, e.g., by a releasablesuture 32. The cannula 28 guides the empty socklet 30 into the injurysite 10. In this arrangement, individual particles 14 (i.e., notconfined during delivery within a mesh socklet 26 as shown in FIG. 9)can be urged through the cannula 28, using, e.g., a tamp, to fill thesocklet 30 within the injury site. Upon filling the socklet 30 withparticles 14, the suture 32 can be pulled to release the cannula 28,leaving the particle filled socklet 30 behind in the injury site 10, asFIG. 11B shows.

Alternatively, as FIG. 12 shows, individual particles 14 can bedelivered to the injury site 10 through a syringe 36. In thisarrangement, means for targeting of the particles 14 at the injury site10 and protection against disbursement of the particles 14 away from theinjury site 10 due to blood flow may be required, using the alreadydescribed confinement devices and techniques. It is believed thatpermanent internal use will require the use of a socklet or equivalentconfinement technique.

II. GRANULAR HEMOSTATIC AGENT Another Embodiment

Granular chitosan salt and potentially granular chitin by itself or incombination with inorganic calcium would be a very useful hemostaticagent for rapid delivery to superficial and difficult to access bleedingsites. Such granules, if prepared from suitable purified stock, wouldalso be able to be used without concern for immunological, inflammatory,cytotoxic or thermal injury effects (due to control of hemostasis byproducing heat).

An effective hemostatic particulate (granular) chitosan foam has beenpreviously described. In an alternative embodiment, as will now bedescribed, an equally effective granular hemostatic form (i.e.,comprising another representative form of the particle 14 as previouslydescribed) can be created with minimal processing, almost directly froma chitosan source supply. In this embodiment, no lyophilization or otherfoam forming procedure is necessary. The granular hemostatic material inthis embodiment is obtained from controlled grinding (to controlledparticle size) of deproteinized and potentially decalcified (orpotentially fully decalcified) crustacean shell material, which is thenpartially (30%) or near fully (80-85%) deacetylized.

Deproteinized and decalcified crustacean exoskeleton is most generallyreferred to as “chitin” or polyβ-(1→4)w-acetyl-D-glucosamine orpolyβ-(1→4)2-acetamide-2-D-glucopyranose. The correct nomenclature(RUPAC) is poly[β-(1→4)-2-acetimide-2-deoxy-D-glucopyranose, however forconvenience we will call this material (deacetylized, to at most 30%)chitin.

The chitin shell material can be obtained, e.g., from squid, crabs, orother crustacean. The chitin granule, as ground to form the particle 14,may be used to control hemorrhage and to act as a hemostatic agent inall the manners and embodiments previously described.

To enhance the surface area of the chitin granule, it may be expandedand partially ruptured by rapid release under vacuum of an absorbed,volatile swelling agent. Such partially ruptured and expanded particles,beads or granules 14 of chitin could be achieved by rapid release ofpressure heating in particles containing a high fraction of absorbed CO₂or other volatile solvent. The extent of rupturing of the particle maybe controlled by the uniformity and depth of absorption of the volatileswelling agent in the particle or granule.

Controlled rupturing of particle surface and bulk results in thepreparation of granules 14 with controlled surface and bulk properties.Freezing of absorbed solvent (one which experiences an increase inspecific volume or freezing) would be another method for controlledrupturing.

As an example, a particle 14 which is prepared with about two-thirds ofthe interior of the particle radius intact (not expanded) and about theother one-third of particle radius expanded at the surface enables adense particle core and a significantly less dense, high specificsurface area particle surface. The high specific area surface providesfor enhanced hemostatic interaction with blood, while the dense coreprovides for sufficient particle density to overcome buoyancy and otherfluid flow related delivery problems.

Enhanced density can also be achieved by the addition of iron and/orcalcium to the expanded particle. Enhanced density leads to enhancedhemostasis.

Chitin or expanded chitin particles 14 may be further processed tochitosan by enzymatic or hydrolytic treatment. Chitosan is generallychitin that has been deacetylated to more than 50% degree ofdeacetylation. Although it generally does contain residual acetyl groupswhich are present in block or random repeat units along the polymerchain, chitosan is often referred to as polyβP-(1→4)D-glucosamine ormore correctly (RUPAC) polyβ-(1→4). w-acetyl-D-glucosamine orpolyβ-(1→4)2-amino-2-deoxy-D-glucopyranose.

Because all commercial deacetylation of chitin is done heterogeneously(particles dispersed in a deacetylation medium) it is possible toprepare particles with a non-uniform degree of deacetylation. Suchnon-uniformity would present itself as a higher degree of deacetylationat the particle surface compared to the particle core. Granules,particles or beads with higher degree of deacetylation at the surfacecompared to the bulk is advantageous in preparation of highlyefficacious hemostatic chitosan/chitin granules since addition of aceticacid to the granular surface or other types of acid such as lactic,glycolic hydrochloric, glutamic, propionic, citric or other mono-acids,di-acids or tri-acids provides for catonic and muco-adhesive propertiesthat promote erythrocyte agglutination and enhanced hemostasis. Theadvantage of surface localized muco-adhesive properties ensures that thereactive groups are located where they will most effectively interactwith blood and also the core of the particle is not muco-adhesive,meaning that it is insoluble in blood and will provide a dense chitincenter for extended efficacy of the particle, for its effective deliveryand for enhanced agglomeration of blood and other particles of similarstructure to form a strong and adhesive clot.

Another advantage of localization of the deacetylated chitin (chitosan)at the particle surface, especially in the case of ruptured/expandedbead or granule is that derivatization of the chitosan amine groups isthen readily possible to create a high specific surface areafunctionalized bead/granule/particle that could be used effectively forhemostasis, antibacterial or antiviral application.

III. CONCLUSION

It should be apparent that above-described embodiments of this inventionare merely descriptive of its principles and are not to be limited. Thescope of this invention instead shall be determined from the scope ofthe following claims, including their equivalents.

The invention claimed is:
 1. A hemostatic chitosan material in granuleor particle form having a density between about 0.25 to 0.60 g/cm³ and agranule or particle diameter of about 0.9 mm, wherein said granules orparticles are produced by freeze-drying a frozen matrix of chitosan,heat-compressing said matrix, and then granulating the compressedmatrix.
 2. The hemostatic chitosan material of claim 1, wherein the atleast one of the granules or particles includes dense chitosan beads. 3.The hemostatic chitosan material of claim 1, wherein the density of thehemostatic chitosan material in the form of a granule or particle isbetween about 0.25 to 0.50 g/cm3.
 4. The hemostatic chitosan material ofclaim 1, further comprising a chitosan salt.
 5. The hemostatic chitosanmaterial of claim 4, wherein the chitosan salt is selected from thegroup consisting of chitosan acetate, chitosan lactate, and chitosanhydroxyacetate.
 6. A hemostatic device comprising a mesh socklet and thehemostatic chitosan material of claim
 1. 7. The hemostatic device ofclaim 6, wherein the device further comprises strips of a polymer meshmaterial.
 8. The hemostatic device of claim 6, wherein the mesh sockletcomprises a polymer material.
 9. The hemostatic device of claim 6,wherein the mesh socklet comprises openings sized to allow for thegranules or particles to protrude out of the mesh socklet.
 10. Thehemostatic device of claim 6, wherein the density of the chitosanmaterial in the form of a granule or particle is between about 0.25 to0.50 g/cm3.
 11. The hemostatic device of claim 6, wherein the chitosanmaterial comprises a chitosan salt.
 12. The hemostatic device of claim11, wherein the chitosan salt is selected from the group consisting ofchitosan acetate, chitosan lactate, and chitosan hydroxyacetate.
 13. Thehemostatic device of claim 6, further comprising a cannula.
 14. Thehemostatic device of claim 6, further comprising a syringe.
 15. Ahemostatic agent comprising chitosan or chitin crustacean shell materialwhich has been at least partially or fully deproteinized, and partiallyor fully deacetylized, wherein the chitosan or crustacean shell materialis ground into granule or particle form, and wherein the chitosan orchitin granules or particles are expanded and partially ruptured suchthat about two-thirds of an interior of a granule or particle radius isintact and about a remaining one-third of the granule or particle radiusis expanded at the surface of said granule or particle.
 16. Thehemostatic agent of claim 15, wherein the granule or particle form isfully deproteinized and partially or fully deacetylized chitosanmaterial.
 17. The hemostatic agent of claim 16, wherein the chitosanmaterial comprises a chitosan salt.
 18. The hemostatic agent of claim17, wherein the chitosan salt is selected from the group consisting ofchitosan acetate, chitosan lactate, and chitosan hydroxyacetate.